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Polypyrimidine Tract Binding Proteins Are Essential for B Cell Development

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bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Polypyrimidine Tract Binding Proteins are essential for B cell development Authors: Elisa Monzón-Casanova1,2*, Louise S. Matheson1, Kristina Tabbada3, Kathi Zarnack4, Christopher W. J. Smith2 and Martin Turner1* Affiliations: 1Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK; 2Department of Biochemistry, University of Cambridge, Cambridge, UK; 3Next Generation Sequencing Facility, The Babraham Institute, Cambridge, UK; 4Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany *Corresponding authors: Elisa Monzón-Casanova ([email protected]) and Martin Turner ([email protected]) 1 bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Abstract During B cell development, recombination of immunoglobulin loci is tightly coordinated with the cell cycle to avoid unwanted rearrangements of other genomic locations. Several factors have been identified that suppress proliferation in late-pre-B cells to allow light chain recombination. By comparison, our knowledge of factors limiting proliferation during heavy chain recombination at the pro-B cell stage is very limited. Here we identify an essential role for the RNA-binding protein Polypyrimidine Tract Binding Protein 1 (PTBP1) in B cell development. Absence of PTBP1 and the paralog PTBP2 results in a complete block in development at the pro-B cell stage. PTBP1 promotes the fidelity of the transcriptome in pro-B cells. In particular, PTBP1 controls a cell cycle mRNA regulon, suppresses entry into S-phase and promotes progression into mitosis. Our results highlight the importance of S-phase entry suppression and post-transcriptional gene expression control by PTBP1 in pro-B cells for proper B cell development. 2 bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Introduction During development B cells first rearrange VDJ gene segments of their immunoglobulin heavy chain (Igh) locus and subsequently VJ gene segments from their immunoglobulin light (Igl) loci to generate antibody diversity (Supplementary Fig. 1a). A burst in proliferation at the early-pre-B cell stage occurs once developing B cells have successfully recombined their Igh locus (Herzog et al., 2009). V(D)J recombination occurs during G0/G1 phase of the cell cycle (Schlissel et al., 1993) and RAG proteins are degraded upon entry into S-phase to suppress V(D)J recombination (Li et al., 1996). Therefore, alternation between proliferative and non-proliferative stages during B cell development is precisely controlled (Herzog et al., 2009; Clark et al., 2014). Several factors have been identified that suppress proliferation in late-pre-B cells and allow Igl recombination including interferon regulatory factors-4 and -8 (Irf4, Irf8) (Lu et al., 2003), Ras (Mandal et al., 2009), Ikaros and Aiolos (Ma et al., 2010), BCL-6 (Nahar et al., 2011), dual specificity tyrosine-regulated kinase 1A (DYRK1A) (Thompson et al., 2015), B cell translocation gene 2 (BTG2) and protein arginine methyl transferase 1 (PRMT1) (Dolezal et al., 2017). By comparison, we are only beginning to understand mechanisms that control proliferation in pro-B cells. At this stage, cell cycle progression driven by IL-7 is segregated from Igh recombination (Johnson et al., 2012) and the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 suppress proliferation allowing Igh recombination and B cell development (Galloway et al., 2016). A network of transcription factors controls development and identity in B cells (Busslinger, 2004). After transcription, RBPs control messenger RNA (mRNA) expression and coordinate functionally related genes in mRNA regulons (Keene, 2007). Control of 3 bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. cell cycle mRNA regulons by RBPs is fundamental for early lymphocyte development (Galloway and Turner, 2017). Polypyrimidine Tract Binding Proteins (PTBP) are RBPs that control alternative splicing, alternative polyadenylation, mRNA stability and translation (Hu et al., 2018; Knoch et al., 2004; 2014). PTBP1 can either increase or decrease mRNA stability by binding to the 3’UTR of transcripts or by modulating alternative splicing of exons that will form transcript isoforms degraded by nonsense- mediated mRNA decay (NMD) (Hu et al., 2018). PTBP1 is expressed ubiquitously. PTBP2 and PTBP3 (formerly ROD1) are highly conserved paralogs of PTBP1 expressed in neurons and hematopoietic cells, respectively. PTBP1 suppresses expression of PTBP2 directly by inducing Ptbp2 exon 10 skipping and mRNA degradation by NMD (Boutz et al., 2007). In germinal centre B cells, PTBP1 promotes proliferation, the c-MYC gene expression program induced upon positive selection and antibody affinity maturation (Monzon-Casanova et al., 2018). PTBP1 and PTBP2 have specific and redundant functions (Spellman et al., 2007; Vuong et al., 2016; Ling et al., 2016). Therefore, expression of PTBP2 in PTBP1-deficient cells compensates for many functions of PTBP1 (Spellman et al., 2007; Vuong et al., 2016; Ling et al., 2016; Monzon-Casanova et al., 2018). To study the roles of PTBP1 controlling post-transcriptional gene expression in B cells, we and others deleted Ptbp1 in pro-B cells and found normal B cell development accompanied by PTBP2 expression (Monzon-Casanova et al., 2018; Sasanuma et al., 2019). Here we addressed the functions of PTBP in B cell development by deleting both Ptbp1 and Ptbp2 in pro-B cells. We show that PTBP1, and in its absence PTBP2, are essential to promote B cell lymphopoiesis. In pro-B cells PTBP1 suppresses entry into S- phase and promotes entry into mitosis after G2-phase. At the molecular level, PTBP1 is 4 bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. necessary to maintain transcriptome fidelity in pro-B cells and is an essential component of a previously unrecognised cell cycle mRNA regulon. 5 bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. Results Redundancy between PTBP1 and PTBP2 for B cell development As both PTBP1 and PTBP3 are expressed in mature B cells (Monzon-Casanova et al., 2018) we determined their expression during defined stages of B cell development in mouse bone marrow (Supplementary Fig. 1a-d). Fluorescence intensity of intracellular PTBP1 staining by flow cytometry was greater in pro- and early-pre-B cells compared to late-pre-B and immature-B cells. However, the isotype control staining for PTBP1 showed the same differences in fluorescence intensity (Supplementary Fig. 1c, d). Therefore, PTBP1 protein expression was similar between the different bone marrow B cell populations. PTBP3 protein detected with a specific monoclonal antibody (Monzon- Casanova et al., 2018) was also relatively constant throughout B cell development (Supplementary Fig. 1c, d). PTBP2 protein was not detected in any of the B cell developmental stages analysed (Supplementary Fig. 1c, d). Thus, PTBP1 and PTBP3, but not PTBP2, are co-expressed throughout B cell development. Conditional deletion of a Ptbp1-floxed allele (Suckale et al., 2011) in pro-B cells with the Cd79acre allele (Cd79acre/+Ptbp1fl/fl mice, denoted here as Ptbp1 single conditional knock- out, P1sKO) did not affect B cell development and resulted in the expression of PTBP2 (Monzon-Casanova et al., 2018). Therefore, we generated a double conditional knock-out (dKO) mouse model where both Ptbp1 and Ptbp2 were deleted in pro-B cells using floxed alleles and Cd79acre (Cd79acre/+Ptbp1fl/flPtbp2fl/fl mice, denoted here as P1P2dKO). Deletion of both Ptbp1 and Ptbp2 in pro-B cells resulted in the absence of mature B cells in the spleen and lymph nodes (Fig. 1a, b and Supplementary Fig. 1e). 6 bioRxiv preprint doi: https://doi.org/10.1101/769141; this version posted September 14, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. PTBPs are required for progression beyond the pro-B cell stage The absence of mature B cells in P1P2dKO mice resulted from a block in B cell development at the pro-B cell stage (Fig. 1c, d and Supplementary Fig. 1a). We confirmed the lack of PTBP proteins in pro-B cells from the conditional KO mice (Supplementary Fig.
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